Apoptosis is a well-orchestrated pathway of physiological cell death, activated through alterations in the expression of growth regulatory/death-inducing genes. The long-term goal of this PI is to understand the molecular pathway for apoptosis, with emphasis on gene regulatory changes. In a pair of sister human leukemic T cell lines, CEM-C7-14 and CEM-C1-15, that are susceptible or refractory, respectively, to glucocorticoid (GC)-evoked apoptosis, we have observed a correlation between expression and dephosphorylation of the transcriptional repressor E4BP4, a homolog of the pro-apoptotic C. elegans ces-2 gene product, and apoptosis. While several other gene products are also implicated in GC-evoked apoptosis of leukemic T cells, in this proposal we will focus on evaluating the relative contribution of E4BP4 in modulating the process, its mode of action, and its relationship to other changes triggered by GCs. Based on new preliminary data and comments from reviewers, the specific aims of this resubmission have been revised. Specific aim 1 will analyze the relevance of E4BP4 dephosphorylation on its ability to repress transcription and modulate apoptosis through reporter gene and apoptosis assays of cells treated with kinase and phosphatase inhibitors. In specific aim 2, the ability of E4BP4 to regulate downstream evolutionary conserved modulators of apoptosis (Slug/Snail, Bim/Puma, and Bcl2/Bcl-xL) will be evaluated through ectopic E4BP4 expression in CEM-C1-15 cells, or siRNA-mediated E4BP4 knock-down in CEM-C7-14 cells. A CE-C1-15 derived clone expressing ectopic mouse E4BP4 has been developed and exhibits sensitivity to GC-evoked apoptosis. Specific aim 3 will study the ability of E4BP4 to modulate GR-dependent transcriptional activation and repression using the GRE-driven luciferase reporter pHH-Luc, and the c-myc promoter-luciferase reporter HBMLuc, respectively. The relationship between E4BP4 and expression of other known modulators of T cell apoptosis, including cyclin D3, p27kip1, c-Jun, c-Myc and BTG1, will be tested in CEM cells manipulated to either over-express or shut off E4BP4 expression. Relevance to Public Health: Aberrant or inadequate apoptosis contributes to diseases such as autoimmunity, immunodeficiency, neurodegeneration, and cancer. Studies proposed here will promote development of strategies to enforce or prevent apoptosis and manage these conditions.